Monday, December 12, 2011

Big and Tall with FGF-2?

Effects of FGF-2 on human adipose tissue derived adult stem cells morphology and chondrogenesis enhancement in Transwell culture.

"We investigated the effects of FGF-2 on hADSC morphology and chondrogenesis in Transwell culture. hADSCs were obtained from patients undergoing elective surgery, and then cultured in expansion medium alone or in the presence of FGF-2 (10ng/ml).  Expression levels of SOX-9, collagen type II, and aggrecan were all significantly increased in hADSCs expanded in presence of FGF-2.  FGF-2 induced a slender morphology, whereas doubling time and trypsinization time decreased. FGF-2 induces hADSCs chondrogenesis."

"Adult MSCs [have] a large cytoplasmic volume when cultured without bFGF, while fetal and adult bone marrow-derived MSCs cultured with bFGF (5 ng/ml) have a smaller fibroblast-like morphology with a spindle-shaped cytoplasm"

"FGF-2 maintains the small cytoplasmic volume and spindle morphology of hADSCs over repeated passages."<-other means of maintaining small cytoplasmic volume and spindle morphology may be useful to increase height too.

This study seems like it could possibly involve an FGF-2 therapy for height growth:

Erythroid promoter confines FGF2 expression to the marrow after hematopoietic stem cell gene therapy and leads to enhanced endosteal bone formation.

"Transplantation of mouse Sca-1(+) hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. erythroid specific promoter, β-globin[this can be used for other compound treatments that we wish to confine to the bone marrow in attempts to increase height], leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1(+) cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation[and possibly height growth given FGF2's pro-chondrogenic capabilities]."

"due to a very short in vivo half-life of FGF2, daily injection of large quantities of FGF2 protein is necessary"

"the MLV-based HSC gene therapy yields FGF2 levels that are ∼100-fold higher than physiological concentrations"

"FGF2 expression led to a 2-fold increase in Bmpr1b, Bmp2 and Bmp4"<-This isn't that much at all.

"strong positive correlation between serum FGF2 and serum Fgf23"

Metabolic activities and chondrogenic differentiation of human mesenchymal stem cells following recombinant adeno-associated virus-mediated gene transfer and overexpression of fibroblast growth factor 2.

"Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated overexpression of human fibroblast growth factor 2 (hFGF-2), a mitogenic factor also known to influence MSC differentiation, upon the proliferative and chondrogenic activities of human MSCs (hMSCs) in a three-dimensional environment that supports chondrogenesis in vitro. Prolonged, significant FGF-2 synthesis was noted in rAAV-hFGF-2-transduced monolayer and aggregate cultures of hMSCs, leading to enhanced, dose-dependent cell proliferation compared with control treatments (rAAV-lacZ transduction and absence of vector administration). Chondrogenic differentiation (proteoglycans, type-II collagen, and SOX9 expression) was successfully achieved in all types of aggregates, without significant difference between conditions. Most remarkably, application of rAAV-hFGF-2 reduced the expression of type-I and type-X collagen, possibly due to increased levels of matrix metalloproteinase-13, a key matrix-degrading enzyme. FGF-2 overexpression also decreased mineralization and the expression of osteogenic markers such as alkaline phosphatase, with diminished levels of RUNX-2, a transcription factor for osteoblast-related genes."

FGF-2 may have inhibitory effects at high levels due to FGFR1 and FGFR3.

Chondrogenic induction of human mesenchymal stem cells using combined growth factors for cartilage tissue engineering., states that FGFR-2 or FGF-6 with TGF-Beta2 are both able to induce chondrogenesis.

FGF-2 abolishes the chondrogenic effect of combined BMP-6 and TGF-beta in human adipose derived stem cells., states that FGF-2 can counteract the effects of BMP-6 and TGF-Beta in chondroinduction(and the mechanism is against BMP-6 not TGF as FGF-2 can promote chondrogenesis in the presence of tgf).  Thus, FGF-2 must be involved in some sort of feedback mechanism that in some circumstances can inhibit chondrogenesis.

"In ASC, it is reported that TGFβ receptor I (TGFβRI) is only expressed in the presence of BMP-6, which might be responsible for the reduced chondrogenic potential compared with BMSC. The chondrogenic potential of BMP-6 in alginate suspended ASC and a 100–350-fold upregulation for aggrecan mRNA and a 20–75-fold increase in collagen II mRNA expression after chondrogenic induction [was observed]. However, a concentration of 500 ng/mL BMP-6 was necessary to achieve the upregulation. In combination with TGF-β3, a concentration of only 10 ng/mL BMP-6 was sufficient to induce collagen type II expression on both the mRNA and protein level."

"BMP signaling acts through c-terminal phosphorylation of Smad1, Smad5 or Smad8. Activated Smad1 binds to the nuclear translocation factor Nup214 and initiates the transcription of chondrogenic genes. In the presence of FGF, MEK1 activates the MAPK pathway, which also phosphorylates Smad1. In this case, phosphorylation takes place at the linker region between MH1 and MH2 of Smad1, differing from the situation in normal BMP signaling. In contrast to c-terminally activated Smad1, linker-phosporylated Smad1 binds Smurf1, leading to polyubiquitination and finally to degradation of Smad1."<-So FGF-2 may degrade Smad1 resulting in an inability of BMP-6 to promote chondrogenesis although this doesn't explain why FGF-2 doesn't now promote chondrogenesis by it's own mechanism.

Fibroblast growth factor 2 enhances the kinetics of mesenchymal stem cell chondrogenesis.

"Treatment of mesenchymal stem cells (MSCs) with fibroblast growth factor 2 (FGF-2) {LSJL upregulates FGF2} during monolayer expansion leads to increased expression of cartilage-related molecules during subsequent pellet chondrogenesis. This may be due to faster differentiation and/or a durable change in phenotype. In order to evaluate changes over time, we assessed chondrogenesis of human MSCs at early and late time points during pellet culture. Marked enhancement of chondrogenesis was seen early compared to controls. However, the differences from controls in gene expression dramatically diminished over time. Depending on conditions, increases in glycosaminoglycan accumulation were maintained."

"FGF-2 may influence at least two aspects of MSC chondrogenesis. First, the growthfactor may speed the sequential, time-dependent pattern of gene expression that occurs during cartilaginous differentiation leading to earlier production of cartilage-associated molecules. Secondly, FGF-2 treatment may lead to an altered phenotypic state"

"Pellets from cells grown with 10 ng/mL of FGF-2 had significantly higher levels of Col II (92-fold), Col X (15-fold), ACAN (18-fold), and SOX9 (10-fold) gene expression compared to controls"

"FGF-2 may select for MSCs with inherent chondrogenic potential during monolayer culture"

"FGF-2 may generally enhance the chondrogenic potential of MSCs (priming mechanism) in part by increasing Sox9 protein levels. Our results support a priming mechanism"

Fibroblast growth factor control of cartilage homeostasis.

"FGF-2 selectively activates FGF receptor 1 (FGFR1){up in LSJL} to exert catabolic effects in human articular chondrocytes and IVD tissue via upregulation of matrix-degrading enzyme production, inhibition of extracellular matrix (ECM) accumulation and proteoglycan synthesis, and clustering of cells characteristic of arthritic states. FGF-18 most likely exerts anabolic effects in human articular chondrocytes by activating the FGFR3 pathway, inducing ECM formation and chondrogenic cell differentiation, and inhibiting cell proliferation. These changes result in dispersed chondrocytes or disc cells surrounded by abundant matrix. The role of FGF-8 has recently been identified as a catabolic mediator in rat and rabbit articular cartilage. The available evidence reveals the promise of FGF-2/FGFR1 antagonists, FGF-18/FGFR3 agonists, and FGF-8 antagonists (ie. anti-FGF-8 antibody) as potential therapies to promote cartilage regeneration."

"FGF-2 is produced endogenously in cartilage and has been proposed to be sequestered by
perlecan, a heparan sulfate proteoglycan (HSPG) localized in the extracellular matrix (ECM) of
articular cartilage. Upon cartilage injury, FGF-2 is released from its bound matrix and subsequently activates the ERK signaling pathway"<-Catabolic effects of FGF2 are mentioned which did not occur in LSJL.

"FGF-2 has been found to activate both FGFR1 and FGFR3, its catabolic activities were recently found to be specifically mediated by FGFR1"

"The binding of FGF-2 to FGFR1 leads to receptor phosphorylation, which in turn activates two critical signaling mediators, Ras and Protein kinase C delta. These molecules then integrate their signaling inputs into the Raf-MEK1/2-ERK1/2 cascade to regulate target gene expression"

"All three mitogen activated protein kinase (MAPK) subgroups (ERK, p38 and JNK){LSJL likely upregulates ERK and p38 but activation of JNK is unclear} converge on the transcription factor Elk-1, which transactivates MMP-13, ultimately promoting cartilage degradation. FGF-2 signaling also results in the activation of AP-1 and RUNX2, the latter of which may account for ADAMTS-5 induction"

In contrast to growth cartilage FGFR3 is anabolic in articular cartilage.

"Local delivery of adenovirus expressing fgf18 into the pinnae of nude mice induced the formation of auricular cartilage, type II collagen, proteoglycan accumulation, and chondrocyte proliferation"

"FGFR3 can both promote and inhibit chondrocyte proliferation depending on the stage
of development. FGF-18 signaling through FGFR3 may enhance chondrocyte proliferation in immature committed chondrocytes, even though it is well established that signaling through FGFR3 inhibits chondrocyte proliferation and differentiation in the mature proliferating chondrocyte zone of the growth plate"

"degradation of the ECM was promoted in the presence of FGF-8 and this degradation was enhanced when combined with interleukin-1 (IL-1). FGF-8 also induced the production of MMP-3 and prostaglandin E2 (PGE2) in rabbit articular chondrocytes"

Src and fibroblast growth factor 2 independently regulate signaling and gene expression induced by experimental injury to intact articular cartilage

"Protein tyrosine phosphorylation occurred within seconds of injury to the surface of intact articular cartilage, as did activation of MAPKs and IKK. Activation did not reoccur upon reinjury of cultured explants. The prominent tyrosine-phosphorylated proteins focal adhesion kinase, paxillin, and cortactin were identified as substrates of Src family kinases. The Src family kinase inhibitor PP2 blocked injury-induced tyrosine phosphorylation. It did not prevent activation of the MAPKs and IKK but differentially inhibited 8 of 10 inflammatory response genes that were induced by injury. In contrast, FGF signaling blockade with PD173074 reduced all MAPK and IKK activation by ∼50% and inhibited a different subset of genes but had no effect on Src-like signaling."

"Both FAK and paxillin are known to be tyrosine phosphorylated, and both were observed only in the lanes showing results for cartilage 10 minutes after dissection"

"Cartilage dissection caused phosphorylation of ATF-2 after 10 minutes, but PP2 caused no reproducible inhibition of this phosphorylation"

Gene expression comparison to LSJL to be done.

Fibroblast growth factor 2 (Fgf2) inhibits differentiation of mesenchymal stem cells by inducing Twist2 and Spry4, blocking extracellular regulated kinase activation, and altering Fgf receptor expression levels.

" fibroblast growth factor 2 (Fgf2) reversibly inhibited multilineage differentiation of primary mouse MSCs and now identify a unique compliment of signaling proteins that are dynamically regulated by this mitogen and whose expression levels are strongly correlated with inhibition of cell differentiation. Fgf2 selectively induced expression of Twist2 and Sprouty4 (Spry4) and repressed expression of soluble frizzled related receptor 2 (Sfrp2), runt-related transcription factor 2 (Runx2), and peroxisome proliferation activated receptor gamma (Pparg). In contrast, Wnt3a induced expression of Twist but not Twist2 or Spry4 and bone morphogenetic protein 2 (Bmp2) failed to alter expression of all three genes. Moreover, pretreatment of MSCs with Fgf2 delayed extracellular regulated kinase 1 (Erk1) and Erk2 phosphorylation and repressed bone-specific gene expression during an osteoinduction time course. Alternatively, pretreatment with Wnt3a had no effect, whereas Bmp2 pretreatment augmented Erk1/2 activation and bone-specific gene expression. Fgf2 also induced expression of Fgf receptor 1 (Fgfr1) and Fgfr4 and repressed Fgfr2 and Fgfr3 expression in MSCs, whereas Wnt3a and Bmp2 had the opposite effect. Twist and Spry4 [are] coexpressed in MSCs and Fgf2 treatment altered their subcellular distribution in a manner consistent with their mode of action. Inhibition of mouse MSC differentiation by Fgf2 is strongly correlated with upregulation of Twist2 and Spry4 and suppression of Erk1/2 activation."

"exogenous application of Wnt3a or ectopic expression of the Lpr5 receptor has been shown to stimulate MSC growth by inducing cyclin D (Ccnd1) and c-Myc expression. "

" Fgf2 upregulated expression of Twist2, Spry4, and Ccnd1 and depressed expression of Sfrp1 to a significant extent when compared with untreated cells. In contrast, Wnt3a stimulated expression of Twist (4.1-fold) and Ctnnb1 (1.4-folds) and downregulated expression of Sfrp1"

FGF-2 addition during expansion of human bone marrow-derived stromal cells alters MSC surface marker distribution and chondrogenic differentiation potential.

"Mesenchymal stromal cells were harvested from bone marrow of six patients and expanded in alpha-MEM or DMEM-LG. Starting in passage 2, 10 ng/ml FGF-2 was administered and non-supplemented media were used as controls. Growth indices were calculated from P0 to P4.  Standard chondrogenic, adipogenic and osteogenic differentiation protocols were applied.
Cell population growth indices were higher for those in FGF-2 supplemented media. Significant differences in surface marker distribution were observed for CD13, CD14, CD49, CD90, CD340 and STRO-1 depending on respective culture conditions. FGF-2 suppressed CD146 expression in both alpha-MEM and DMEM-LG. No differences in adipogenic and osteogenic differentiation potential could be observed, while FGF-2 significantly improved chondrogenic differentiation in DMEM-LG."

"acceleration of proliferation from the beginning of FGF-2 administration, and improved chondrogenic outcome for FGF-2 augmented groups."<-FGF2 may enhance MSC proliferation and therefore enhance mesenchymal condensation.

1 comment:

  1. Is there no way to increase FGF2